Antibodies Against Kchlp2 Induce Ionic Imbalance And Cell Death In Isolated Rat Cardiomyocytes

Disturbances of humoral immunity have been described in patients with dilated cardiomyopathy (DCM). Antibodies against Kv channel-interacting proteins (KChIPs) may be associated with heart failure. Isolated rat cardiomyocytes were treated with antibodies against rat KChIP2 (80 pmol/ml) up to 24 hours. RNA and proteins were isolated after two hours by standard procedures and mRNA (TaqMan®) and protein (Western blot) expression was quantified. Translocations of NF-κB subunits p50, p65, c-Rel, and Rel-B were measured in nuclear protein extracts by ELISA after 60 minutes. Mitochondrial membrane potential ΔΨm and caspase-3 and -9 activities were determined by flow cytometry. Necrotic and apoptotic cells were distinguished by staining with Hoechst 33258 and propidium iodide. Total Ca 2+ and K + concentrations were quantified by a colorimetric assay and atomic absorption spectrometry, respectively, and normalized to the protein content of the cells. Antibodies against KChIP2 induced nuclear translocation of all NF-κB subunits analyzed. Pre-incubation with a blocking peptide or an NF-κB inhibitor, CAPE, prevented nuclear translocation. Anti-KChIP2-treatment for two hours significantly reduced KChIP2 mRNA (55±10%; n=4) and protein (73 ±5%, n =4) expression compared to cells treated with experimental buffer (100%). Treatment for 24 hours did not induce changes in mitochondrial membrane potential, ΔΨm. Caspase-3 and -9 activities were not altered as well. The anti-KChIP2-treated cell population consisted of 75±3% necrotic, 2±1% apoptotic, and 13±2% viable cells. In contrast, cells treated with experimental buffer were viable to 86±1%. After five minutes, anti-KChIP2 induced significant increases in total intracellular Ca 2+ (plus 11±2%) and K + (plus 18±2%) concentrations. These antibody-mediated effects were prevented in the presence of a blocking peptide. Antibodies against KChIP2 induce ionic imbalance, activate the transcription factor NF-κB, down-regulate KChIP2 expression and enhance cell death rate probably due to necrosis. Antibodies against KChIP2 may contribute to the development and progression of dilated cardiomyopathy.